Thrombin is a proteolytic enzyme having multiple functions in blood coagulation. Thrombin is formed from prothrombin (coagulation Factor II), a circulating zymogen precursor protein in the plasma. It is proteolytically cleaved to form thrombin in the coagulation cascade. Thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions.
Thrombin is widely used in clinical applications as a coagulation factor to staunch bleeding of wounds by conversion of fibrinogen to fibrin. It is a common component of surgical dressings, and has been used in combination with fibrinogen and other coagulation proteins in two-component hemostatic systems such as fibrin glues, adhesives, and sealants.
Thrombin powder for use in preparation of pharmaceutical compositions is commonly prepared by lyophilization of a solution.
The term “lyophilization” typically refers to the process of freezing a solution and then reducing the concentration of water e.g. by sublimation to levels which do not support biological or chemical reactions.
Lyophilization is a batch process which is limited by the capacity of the lyophilizer and usually lasts for several days. This puts the entire drying batch at risk for a significant amount of time until the process is completed. Furthermore, processing the resulting lyophilized “cake” into powder requires executing additional steps e.g. breaking, sieving and/or milling to further reduce particle size. These steps may decrease the potency and mass yield of the dry product. Advantageously, using a spray-drying process instead of lyophilization provides a cost effective, continuous and high-capacity process, in which the dry product is immediately obtained in a form of dry powder and not as a “cake”.
As used herein, the term “cake” or “solid cake” refers to a porous and spongy structure-like composition resulting from the lyophilization process.
Spray drying involves atomization of a liquid, such as a solution, suspension or emulsion, for example by spraying through a spray nozzle while contacting with an atomizing gas to form spray particles; by two-fluid nozzle atomization, wherein spray is created by combination of a liquid flow and a gas flow, in which the atomization energy is provided by the gas flow; or by centrifugal atomization, wherein the solution is delivered in a rotating disk, such that spray is created by the energy created by the rotation of the disc. Alternatively, the liquid flow may be sprayed using a pressure nozzle in which the liquid flow is forced through a small aperture, the change in pressure transforming the liquid flow into spray of small droplets.
Formation of spray particles is followed by drying of the spray in a flow of a hot gas (e.g. air or nitrogen) provided by a drying gas conduit. The spray particles dry rapidly into powder, which is then separated from the hot airflow in a cyclone device, and may be collected in a container e.g. a vial. The spray drying process is controlled by several key process parameters, including drying air flow rate and temperature, atomizing air flow rate, solution flow rate and more.
Generally, enzymes such as thrombin are sensitive to the high temperatures and sheer stress conditions used in prior art spray drying processes, which usually cause denaturation and subsequent loss of potency of the enzyme. For example, thrombin in solution is sensitive to temperatures of about 45° C. or above.
The prior art solves this problem by using relatively low thrombin concentrations in the solution used for spray drying, or by increasing the concentration of carbohydrates, such as trehalose in the solution. These conditions result in a spray dried powder comprising a low concentration of thrombin (generally up to about 1 IU/mg).
Examples of background art spray drying processes for production of thrombin powder and uses thereof include U.S. Pat. No. 6,113,948; U.S. Publication No. 2012/0315305; PCT Publication No. WO 92/18164; U.S. Pat. No. 6,416,739; European Patent No. EP0713388B1; U.S. Pat. No. 8,846,105; U.S. Pat. No. 6,703,047; U.S Publication No. 20120121532; U.S. Publication No. 20100249044; PCT Publication No. WO 14/135689; J Microencapsul. 2013; 30(7):624-31. Doi: 10.3109/02652048.2013.770097. Epub Mar. 14 2013; Biotechnol Appl Biochem. 1995 October; 22 (Pt 2):203-14; and Excipient Mediated Biostabilization of Protein Using Spray Drying Technique—Thesis work done by Priyadarsini Pattnayak for a degree of master of technology, Biotechnology and medical engineering, under the guidance of Prof. Gyana Ranjan Satpathy, National institute of technology, Rourkela, India. September 2010.